1. Field of the Invention
The present invention relates to nucleic acid sequences that encode polypeptides targeted to the peroxisome, wherein the polypeptides have protease activity and process peroxisomal enzymes. In a particular embodiment, the invention provides proof that the peroxisomal protease Tysnd1 is capable of processing PTS1- and PTS2-signal containing enzymes Acaa1, Acox1, Scp2, and Hsd17b4 involved in the peroxisomal β- and α-oxidation. The invention further relates to therapeutic, diagnostic and research methods for diagnosis, treatment, and prevention of disorders involving any one of these mammalian nucleic acids encoding peroxisomal proteases.
2. Description of the Related Art
Peroxisomes are organelles present in all eukaryotic organisms studied so far. Since peroxisomes lack DNA and protein synthesis capabilities, all peroxisomal proteins are synthesized in the cytosolic compartment and post-translationally sorted to the peroxisome [1-2]. Two distinct peroxisomal signal targeting sequences (PTS) and their variants, the C-terminal PTS1 and the N-terminal PTS2, have been defined. Almost all peroxisomal enzymes have the PTS1 signal [SA]-K-L which was subsequently expanded to [STAGQCN]-[KRH]-[LIVMAFY][3] and this PTS1 signal is recognized by the cytosolic soluble receptor Pex5p. Only a few peroxisomal proteins are targeted via the N-terminally located PTS2 motif [RK]-[LVI]-[X5]-[HQ]-[LAF][4-6]. A small number of peroxisomal matrix proteins that lack both PTS1 and PTS2 signals are targeted to the organelle by poorly defined internal PTSs [7].
The function of peroxisomes is extremely diverse and dependent on the cell type and external stimuli. In humans peroxisomes are involved in a variety of anabolic and catabolic pathways (e.g. cholesterol biosynthesis, fatty acid oxidation, purine metabolism, hydrogen peroxide detoxification, bile acid synthesis, plasmalogen synthesis, amino acid metabolism) [8-9], infectivity of human immunodeficiency virus and rotavirus [10] and certain developmental processes that are independent of the metabolic state [11]. In yeast, peroxisomes are essential for the metabolism of unusual carbon sources such as oleic acid, primary amines, purines, D-amino acids, and methanol [12-13]. In plants, peroxisomes are involved in photorespiration [14], in trypanosomes peroxisomes are involved in glycolysis [15-16], and in fungi they are involved in the synthesis of secondary metabolites, for example beta-lactam penicillins [17].
Mass spectrometry (MS), in combination with the rapid development of sequence databases has significantly enhanced the global characterization of the peroxisomal protein composition in model organisms. The MS-based methodology allowed the identification of 34 known and five putative peroxisomal proteins from rat liver [18] Kikuchi et al 2004). Several genes are involved in the production of peroxisomes in a cell, which is also termed peroxisomal biogenesis. So-called PEX genes encoding peroxins, have been cloned by the functional complementation of yeast mutant strains lacking functional peroxisomes [7]. Thirteen human PEX homologues have been identified through sequence database screening, of which 11 were shown to restore peroxisome biogenesis in cell lines of patients with peroxisomal disorders [19]. Defects in peroxisomal biogenesis contribute to several inherited human disorders, such as Refsum's disease [20], X-linked adrenoleukodystrophy (X-ALD), mevalonic aciduria [21] among other metabolic diseases.
The import of peroxisomal proteins does not seem to involve significant protein modifications [22]. Folded polypeptides and protein dimers can be imported into the peroxisomal matrix [23-24]. Alcohol oxidase monomers are imported into the matrix before the assembly of enzymatically active octamers [25-26], and alanine: glyoxylate aminotransferase 1 can be imported with equal efficiency as a dimer or monomer [24]. Two peroxisomal import models have been proposed. One model proposes that peroxisomal import receptors are shuttling between the cytosol and the peroxisome [27-29]. The other model suggests that peroxisomal receptors pull proteins into the peroxisome [30-31].
However little is known about the fate of proteins with regard to processing, activation, degradation and associated diseases thereof once they have entered the peroxisome. So far two proteases, insulin-degrading enzyme (IDE) [32] and a peroxisome-specific form of Lon protease [18], have been experimentally detected in peroxisomes. IDE may play a role in degradation of oxidized peroxisomal lysozymes while the novel LON protease might be involved in peroxisome biogenesis.
Kurochkin et al. [33-35] computationally identified in a search of 130629 putative translations of GenBank 139.0 rodent and primate mRNA sequences 29 novel peroxisome PTS1-targeted protein candidates. One of the candidates 1300019N10Rik, which is now called Tysnd1 or trypsin domain containing 1 (GenBank and GenPept accessions AK005069 and BAB23793) and its orthologs in rat (XM—345106 and XP—345107), and human (NM—173555 and NP—775826) are weakly similar to a protease-related protein derived from Arabidopsis thaliana. It also contains two protease-related domains, glutamyl endopeptidase I (IPR008256) and trypsin-like serine and cysteine proteases (IPR009003). The members of the glutamyl endopeptidase I family of proteases possess serine-type peptidase activity. Proteolytic enzymes that exploit serine in their catalytic activities are ubiquitous, being found in viruses, bacteria and eukaryotes. The peroxisome is likely to recruit a wide spectrum of proteases, each with a unique specificity, to achieve efficient breakdown of proteins in the organelle.
Tysnd1 is located on mouse chromosome 10. Its human ortholog maps in syntenic position to chromosome 10. Tysnd1 is expressed in adipose tissue, aorta, liver, kidney and lung (see Table 1). Co-expressed genes include Peci, Pex6, Pex16, which are known to encode peroxisome-targeted gene products. Other co-expressed genes (e.g. Fsp27 and Cas1) are associated with fat metabolism. The data derived from public gene expression resources (GNF U74A, GNF Atlas 2, Mouse Gene Prediction Database) suggest that Tysnd1 is involved in peroxisome-regulated fat metabolism.
TABLE 1Tysnd1 gene structure, motifs, expression patternsMouse Tysnd1Human TYSND1Chromosome and10, +strand [36]10; −strand [37]orientationPosition61, 457, 382-61, 464, 634 (NCBI 3371, 568, 974-71, 575, 956 (NCBI 35 genomegenome assembly) [36]assembly) [37]Exon no4 [36]2-4 [37]Neighboring geneupstream: Sara1upstream: AMIDdownstream: Amid [36]downstream: SARA1 [37]TranscriptAK005069, AW121748, AW490206,BC016840, BC030242, BC042629, BC047424informationBB224225 (GenBank) [38](GenBank) [39]ProteinBAB23793 568 aa (GenPept);AAH16840 435 aa, AAH30242 398 aa,informationQ9DBA6 568 aaAAH42629 399 aa (GenPept), Q96AR(SwissProt/TrEBML) [38, 40](SwissProt/TrEMBL) 435 aa, Q8IVQ3399aa, Q5SQU1 398 aa, Q5SQT4 566 aa[39, 40]InterPro motifsPeptidase, trypsin-like serine andPeptidase, trypsin-like serine and cysteinecysteine protease; positions 27-42,potease; positions 54-162 and 222-398 of187-294 and 308-531 of Q9DBA6Q96AR5; 289-534 of Q5SQT4; 309-373 of[41]Q5SQU1 [41]ExpressionKidney, liver, lung, adipose tissue,Testis, lung, liver, adipocytes, skin, thymusaorta, brown fat [42, 43][44]Co-expressedPex16, Pex6, Peci, Cas1, Fsp27,ACAT1, ADH5, COX10 homolog and othersgenesAmid, Scp2, Acaa1, and others[44, 45][42, 43]